Abstract 131: ALK oncogene regulates epithelial-mesenchymal transition (EMT) in ALK-rearranged non-small cell lung carcinoma through repression of the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2)

A subset of Non-Small Cell Lung Carcinoma (NSCLC) carries chromosomal rearrangements involving the Anaplastic Lymphoma Kinase (ALK) gene. More frequently ALK is juxtaposed to the echinoderm microtubule-associated protein-like 4 (EML4) gene on chromosome 2 and generates a constitutively active EML4-ALK fusion protein that triggers downstream oncogenic signals leading to increased cell proliferation and survival. The majority of ALK-rearranged NSCLC presents a peculiar histology characterized by a solid signet-ring cell and a mucinous cribriform pattern that is frequently associated with a metastatic phenotype. As the signet ring phenotype and metastasis are associated with epithelial-mesenchymal transition (EMT), a cellular reprogramming often activated in cancer cells during invasion and metastasis, we investigated whether ALK induces EMT in NSCLC. We performed RNA sequencing analysis on human ALK-rearranged NSCLC cell lines treated with ALK inhibitors or where EML4-ALK was knocked-down by shRNA. We found that EML-ALK regulated several genes related to EMT. In particular, the epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2), key regulators of a splicing switch during EMT, were repressed in ALK-rearranged NSCLC cells and their repression was dependent on ALK activity. In keeping with these observations, H2228 and DFCI032 cells displayed a mesenchymal phenotype with almost complete suppression of the epithelial marker, E-cadherin, and a strong expression of the mesenchymal markers, vimentin and N-cadherin. In H2228 and DFCI032 cells, both E-cadherin suppression and vimentin up-regulation were dependent upon EML-ALK kinase activity because treatment with ALK inhibitors (TAE684 and crizotinib) or ALK knock-down reverted the phenotype of H2228 and DFCI032 from mesenchymal to epithelial and decreased their invasive potential. We excluded the involvement in EMT of ALK-rearranged NSCLC of other RTKs, such as EGFR or MET, because their inhibition did not have any effect on E-cadherin and vimentin expression. In ALK-rearranged NSCLC, the regulation of E-cadherin suppression was mainly transcriptional whereas vimentin regulation was post-transcriptional for both cell lines. Overexpression of ESRP1 led to up-regulation of E-cadherin, whereas ESRP1 knock-down impaired the reversion to an epithelial phenotype associated to inhibition of ALK activity. In conclusion, we showed that oncogenic ALK regulates EMT in NSCLC through ESRP repression. These findin